Microbiology (2004), 150, 22292235

DOI 10.1099/mic.0.27135-0

Symbionts of the gut agellate Staurojoenina sp. from Neotermes cubanus represent a novel, termite-associated lineage of Bacteroidales: description of Candidatus Vestibaculum illigatum
Ulrich Stingl,1 Annelie Maass,2 Renate Radek2 and Andreas Brune1,3
Correspondence Andreas Brune brune@staff.uni-marburg.de
1

LS Mikrobielle Okologie, Fachbereich Biologie, Universitat Konstanz, 78457 Konstanz, Germany

Institut fur Biologie/Zoologie, AG Protozoologie, Freie Universitat Berlin, Konigin-Luise-Str. 13, 14195 Berlin, Germany Max-Planck-Institut fur terrestrische Mikrobiologie, Karl-von-Frisch-Strasse, 35043 Marburg, Germany

Received 4 March 2004 Revised 21 April 2004 Accepted 27 April 2004

The symbioses between cellulose-degrading agellates and bacteria are one of the most fascinating phenomena in the complex micro-ecosystem found in the hindgut of lower termites. However, little is known about the identity of the symbionts. One example is the epibiotic bacteria colonizing the surface of hypermastigote protists of the genus Staurojoenina. By using scanning electron microscopy, it was shown that the whole surface of Staurojoenina sp. from the termite Neotermes cubanus is densely covered with long rod-shaped bacteria of uniform size and morphology. PCR amplication of 16S rRNA genes from isolated protozoa and subsequent cloning yielded a uniform collection of clones with virtually identical sequences. Phylogenetic analysis placed them as a new lineage among the Bacteroidales, only distantly related to other uncultivated bacteria in the hindgut of other termites, including an epibiont of the agellate Mixotricha paradoxa. The closest cultivated relative was Tannerella forsythensis (<85 % sequence identity). Fluorescence in situ hybridization with a newly designed clone-specic oligonucleotide probe conrmed that these sequences belong to the rod-shaped epibionts of Staurojoenina sp. Transmission electron microscopy conrmed the presence of a Gram-negative cell wall and revealed special attachment sites for the symbionts on the cell envelope of the agellate host. Based on the isolated phylogenetic position and the specic association with the surface of Staurojoenina sp., we propose to classify this new taxon of Bacteroidales under the provisional name Candidatus Vestibaculum illigatum.

INTRODUCTION
Wood-feeding termites depend on their symbiotic gut microbiota for lignocellulose digestion (Brune, 2003). In their guts, an impressively dense assemblage of unusual agellate protists and prokaryotic symbionts contribute to the digestion of their chemically recalcitrant diet (Breznak & Brune, 1994; Radek, 1999; Inoue et al., 2000). Although the role of the agellates in cellulose degradation was discovered almost 80 years ago (Cleveland, 1926), and the fermentative nature of these symbionts had already been outlined in the 1940s (for a review, see Hungate, 1955), many details still remain to be resolved. In particular, the nature and function of the prokaryotic epibionts associated with most of the agellates (e.g. Ball, 1969; Lavette, 1969;
The GenBank/EMBL/DDBJ accession number for the sequence reported in this paper is AY540335.

Starr, 1975; Dolan, 2001) is far from clear. The presence of special attachment sites on the cell envelope of the agellates (Smith & Arnott, 1974; Tamm, 1980; Radek et al., 1992, 1996) indicate tight interactions, but only in the case of Mixotricha paradoxa (Cleveland & Grimstone, 1964) and a devescovinid agellate (Tamm, 1982), has a function of the epibionts in the motility of the host cell been demonstrated. Apart from some epibiotic spirochaetes (Iida et al., 2000; Noda et al., 2003) of several other gut agellates, the epibionts of M. paradoxa (Wenzel et al., 2003) are also among the few instances where the phylogenetic afliation of the prokaryotic partner has been substantiated (Dolan, 2001). Flagellates of the genus Staurojoenina are found only among the dry-wood termites (family Kalotermitidae) (Yamin, 1979; Dolan & Margulis, 1997). In their paper, Dolan & Margulis (1997) published excellent electron micrographs
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U. Stingl and others the protocol of Henckel et al. (1999) and using 1 ml of the extracted DNA as template. After purication (EZNA Cycle-Pure kit, peqlab, Erlangen, Germany), the PCR products (3 ml) were cloned in Escherichia coli, using the TA cloning kit (Invitrogen). Clones with correct-size inserts were sorted by RFLP analysis (Stingl & Brune, 2003).
Sequencing and phylogenetic analysis. The inserts of three

prepared by the late David Chase, which show that the surface of Staurojoenina assimilis from Incisitermes minor is covered with unusual epibiotic bacteria. The ultrastructure of the junctional complexes is completely different from that of the motility symbionts of Caduceia versatilis (Tamm, 1980; dAmbrosio et al., 1999), and only remotely resembles the situation of the second epibiont of M. paradoxa (Cleveland & Grimstone, 1964; Wenzel et al., 2003). It is likely that also the nature of the association between Staurojoenina species and their epibiotic bacteria differs from those examples. In this study, we present a detailed analysis of the identity, phylogenetic position and ultrastructure of the epibionts associated with a hitherto undescribed Staurojoenina species colonizing the gut of Neotermes cubanus, and classify them in a provisional Candidatus taxon.

randomly chosen clones were sequenced on both strands using primers 27F (Edwards et al., 1989), 533F, 907R (Lane et al., 1985; used also reverse complementary), and 1492R (Weisburg et al., 1991) by GATC (Konstanz, Germany). Sequences were checked and assembled using DNAStar software (http://www.dnastar.com). Phylogenetic analysis was done as described elsewhere (Stingl & Brune, 2003), using the ARB program package (http://www. arb-home.de; Ludwig & Strunk, 1996).
Whole-cell in situ hybridization. Gut contents of ve termites were suspended in 900 ml PBS and xed for 12 h using 3?7 % formaldehyde. Hybridization procedure and conditions were as described in Stingl & Brune (2003). The oligonucleotide probe Bac303, designed to detect the Bacteroides/Porphyromonas subgroup of the CFB phylum (Manz et al., 1996), was modied to achieve specicity for the sequences obtained in this study. Probe Sym_Stau_303: 59-CCG GTG TGG GGG ACC TTC-39 had at least one mismatch to all other sequences available in GenBank (www.ncbi.nlm.nih.gov). Maximal formamide concentration for successful hybridization was 15 %, which was used routinely. Non-specic binding of the probes was excluded by checking every sample also with a nonsense probe (Wallner et al., 1993). The information on probe Sym_Stau_303 has been submitted to probeBase (http://www.microbial-ecology.de/probebase; Loy et al., 2003).

METHODS
Termites. A colony of Neotermes cubanus (Freytaud), originally collected in Topes de Collantes, Cuba, was provided by Dr I. Hrdy, Praha. The species identication was conrmed by Drs J. Krecek, Fort Lauderdale, and K. Krishna, New York. Termites were maintained in polyethylene containers on a diet of pine wood and water. Scanning and transmission electron microscopy. For scanning

electron microscopy, worker larvae of N. cubanus were dissected, and the contents of the hindgut paunch were released into 0?2 M sodium phosphate buffer (pH 7?0) containing 2?5 % glutaraldehyde and 4 % OsO4. The samples were xed for 1 h on ice, washed three times in the same buffer, dehydrated in a series of ethanol, and critical-point dried with a Bal-Tec CPD 030. Prior to the investigation with a Philips SEM 515 or a Fei Quanta scanning electron microscope, the samples were sputtered with gold in a Balzers Union SCD 040. For transmission electron microscopy, the agellates were pre-xed for 1 h in 0?05 M sodium cacodylate buffer (pH 7?0) containing 2?5 % glutaraldehyde, washed three times in the same buffer, and post-xed in reduced OsO4 (a fresh 1 : 1 mixture of 2 % OsO4 and 3 % K4[Fe(CN)6]) for 1 h on ice. After several further rinses in buffer, the cells were embedded in 3 % agar, dehydrated in a series of ethanol, and embedded in Spurrs resin. Ultrathin sections were stained with saturated aqueous uranyl acetate for 30 min, followed by lead-citrate staining according to Reynolds (1963), and observed using a Philips EM 208 electron microscope.
Preparation of agellates for DNA extraction. Termite hind-

RESULTS
Electron microscopy Scanning electron microscopy (SEM) showed the presence of rod-shaped bacteria covering the whole cell surface of Staurojoenina sp. (Fig. 1A). Groups of 510 bacterial cells, oriented in parallel to each other, were arranged in oblique or rectangular position. The total number of epibionts per agellate was estimated to be approximately 3500. While the diameter of the cells (0?3 mm) was fairly constant, the length of the symbionts ranged between 2?5 and 6 mm (mean 4?0 mm), indicating asynchronous growth and cell division. Spirochaetal cells were occasionally attached between the rod-shaped epibionts, albeit in much smaller numbers (2090 per agellate, Fig. 1B). Transmission electron microscopy (TEM) of ultrathin sections of the agellates revealed the Gram-negative cell wall of the epibiotic rods (Fig. 1C). In addition to the inner and outer membranes, the cells were surrounded by a diffuse outer layer (capsule). A plaque of electron-dense extracellular material connected the bacteria to the agellate surface. The attachment sites of the agellates had the form of elongated ridges, and electron-dense material below the attachment sites apparently serves to support the plasma membrane. Bacterial rods were found not only attached to the surface of the agellates, but also in vacuoles (Fig. 1D). The vacuoles often showed remnants of the
Microbiology 150

guts were carefully ruptured, and a suspension of gut agellates was prepared as described previously (Stingl & Brune, 2003), except that the cells were not xed with formaldehyde. An aliquot (15 ml) of the suspension was spotted into the rst well of a 10-well Teon slide (Roth). Using an inverted microscope, 5060 unambiguously identied agellates of the genus Staurojoenina, which were identied by their characteristic morphology (Fig. 1A), were aspirated from the suspension with a micropipette and transferred to a well containing 15 ml sterile PBS (Stingl & Brune, 2003). This procedure was repeated twice to minimize the amount of loosely attached bacteria in the sample. Finally, 3040 agellates were transferred to a sterile Eppendorf tube with 200 ml PBS. DNA was extracted with the NucleoSpin kit (Macherey-Nagel), according to the manufacturers instructions, and eluted in 50 ml sterile water.
Cloning of 16S rRNA genes. PCR with primers 27F (Edwards et al., 1989) and 1492R (Weisburg et al., 1991) was performed with a Mastergradient thermocycler and the MasterTaq DNA polymerase kit (both Eppendorf) in a total reaction volume of 50 ml, following

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Fig. 1. Scanning (A, B) and transmission (C, D) electron micrographs of Staurojoenina sp. from Neotermes cubanus. (A) Overview of a Staurojoenina cell, showing two of the four agellar tufts (f), numerous bacterial rods (b), and occasional spirochaetes (s) attached to the surface. (B) Close-up of the cell surface, showing single spirochaetes between the ectobiotic rods. Arrows point to early stages of cell division. (C) Cross-section of the epibiotic rods. In addition to inner membrane (im) and outer membrane (om), the cell is surrounded by a diffuse layer (dl). Electron-dense material supports the plasma membrane of the agellate below the attachment sites (arrowhead). The arrow points to a phagocytized rod-shaped bacterium with remnants of attachment complex. (D) Longitudinal section showing bacteria attached to the cell surface (b) and phagocytized bacteria (arrows).

attachment structures, which conrmed that the bacteria were taken up by phagocytosis. Although phagocytosis apparently occurs all over the non-agellated surface, the highest density of vacuoles was observed in the posterior region of the host cell. While the bacteria in the tightly tting vacuoles located close to the agellate surface appeared fairly intact, those in loosely tting vacuoles located deeper within the agellate cell had a changed morphology and were apparently subject to degradation (not shown). Fusion with lysosomes remains to be demonstrated. Cloning and phylogenetic analysis PCR amplication and cloning of 16S rRNA genes from DNA extracted from a suspension of hand-picked Staurojoenina cells and subsequent RFLP analysis indicated that the resulting clone library contained only a single ribotype. Almost full-length sequences (14171418 bp) were obtained for three randomly chosen clones; since the sequences were
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virtually identical (>99?5 % sequence identity), the clone library was considered homogeneous, and only one sequence was submitted to GenBank. The preliminary BLAST searches (Altschul et al., 1997) had already indicated an afliation of the clones with the Bacteroidales. In a detailed phylogenetic analysis (Fig. 2), the clone from Staurojoenina sp. always fell within a cluster of clones comprising Bacteroides-related sequences from other termites (Cluster 4; Ohkuma et al., 2002), which also included the rodshaped symbiont of the trichomonad agellate Mixotricha paradoxa (Wenzel et al., 2003). The closest cultivated relative was the human oral bacterium Tannerella forsythensis, although the sequence similarity was rather low (<85 %). Assignment of clonal isolates to morphotype by uorescence in situ hybridization (FISH) When hindgut suspensions were hybridized with the bacteria probe EUB 338 (Amann et al., 1990), most prokaryotic
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Fig. 2. Phylogenetic tree of SSU rRNA gene sequences of the symbiotic bacteria associated with Staurojoenina sp. from Neotermes cubanus (marked in bold) and their closest relatives, showing also their relationship to the Bacteroides-related sequences from other termites (Clusters 15, Ohkuma et al., 2002). GenBank accession numbers are included in the tree, which was constructed with the maximum-likelihood algorithm implemented in ARB (fast DNAml; Olsen et al., 1994) and is based on 850 unambiguously aligned sequence positions. Bar, 10 substitutions per 100 nt.

cells, including those attached to the surface of Staurojoenina sp., were labelled (details not shown). By contrast, the sequence-specic oligonucleotide probe Sym_Stau_303, specically designed to detect the 16S rRNA gene sequence of the clones obtained in this study, hybridized exclusively with all rod-shaped bacterial cells colonizing the surface of this agellate (Fig. 3). The probe did not hybridize with any

bacteria located on or within other agellate species or with those freely suspended in the hindgut uid.

DISCUSSION
The results of this study show that the cell surface of the Staurojoenina sp. colonizing the gut of Neotermes cubanus is

Fig. 3. Phase-contrast photomicrographs (A, C) and corresponding epiuorescence photomicrographs (B, D) of Neotermes cubanus hindgut suspensions hybridized with the Cy3-labelled nonsense probe (B) or the sequence-specic oligonucleotide probe Sym_Stau_303 (D). Each image pair shows the same eld of view containing several gut agellates, each including a large Staurojoenina cell. Epiuorescence photomicrographs were taken with the same lter sets and exposure time to illustrate the specicity of probe Sym_Stau_303 for the epibionts of Staurojoenina sp.; the slight autouorescence present in all agellates was not caused by the probes. Bars, 50 mm. 2232 Microbiology 150

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completely covered with rod-shaped micro-organisms. Ultrastructure of the epibionts and their attachment sites closely resembles that of the epibionts of Staurojoenina assimilis from Incisitermes minor (Dolan & Margulis, 1997). The epibionts represent a homogeneous population related to the Bacteroidales, where they form a distinct phylogenetic lineage among the 16S rRNA gene clones of other hitherto uncultivated bacteria from the hindguts of various termites, only distantly related to Tannerella forsythensis [formerly Bacteroides forsythus (Tanner et al., 1986); see also Maiden et al. (2003)]. Although the 16S rRNA genes of bacteria related to the Bacteroidales are regularly encountered in termite guts (Schultz & Breznak, 1978; Ohkuma & Kudo, 1996; Kudo et al., 1998; Berchtold et al., 1999), most of them belong to lineages containing no cultured representatives (Ohkuma et al., 2002; Hongoh et al., 2003; Schmitt-Wagner et al., 2003). Considering the results of the present study, one reason for these cultivation problems might lie in the symbiotic association with agellates and a possibly obligate need for a specic host. Interestingly, the epibionts of Staurojoenina sp. fall into the same cluster of the Bacteroidales as one of the two epibionts of the trichomonad agellate Mixotricha paradoxa from Mastotermes darwiniensis (Wenzel et al., 2003). There are two preliminary reports on the ectosymbionts of the hypermastigid agellate Barbulanympha spp. from the gut of the wood-eating roach Cryptocercus punctulatus (Merritt et al., 1996) and a devescovinid agellate Caduceia species from the gut of Cryptotermes cavifrons (Goss et al., 2000), which were reportedly identied as members of the Bacteroides/Porphyromonas subgroup. Although details were never published, it seems likely that many of the Bacteroidesrelated 16S rRNA genes recovered from the guts of lower termites (Ohkuma et al., 2002; Hongoh et al., 2003) represent epibionts of gut agellates. A larger dataset might allow an excellent case study of co-evolution between the bacterial symbionts and their agellate hosts. The function of the epibionts of Staurojoenina species is not yet clear, but in view of the enormous numbers of bacteria present on each agellate, it is suggestive that they play an important role for the host. Cleveland & Grimstone (1964) provided an elegant description of the fascinating motility symbiosis between Mixotricha paradoxa and its epibiotic spirochaetes, which were recently identied as members of the Treponema cluster by 16S rRNA gene sequence analysis (Wenzel et al., 2003). Mixotricha paradoxa, which occurs only in the gut of Mastotermes darwiniensis, is propelled by the helical undulations of the spirochaetes adhering to the host membrane through specialized cell junctions. The spirochaetes are attached to projecting brackets of the cell surface in a manner that allows the helical movement of the individual cells to travel in metachronal waves along the cell surface of the host, resulting in locomotion (Cleveland & Grimstone, 1964).
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Also the devescovinid agellate Rubberneckia, recently described as Caduceia versatilis (dAmbrosio et al., 1999), is densely colonized by two different bacterial epibionts (Tamm, 1982). In this case, the rod-shaped bacteria (20003000 per agellate) are deeply embedded into the cell surface of the host, which is propelled by the selfsynchronizing movement of the bacterial agella (Tamm, 1982). Although the ultrastructure of the junctional complex is completely different, the morphology of these symbionts resembles that of the epibionts of Staurojoenina. This agrees with the preliminary report that Caduceia spp. from Cryptotermes cavifrons are associated with members of the Bacteroides/Porphyromonas subgroup (Goss et al., 2000). However, our light-microscopy observation of live preparations of Staurojoenina agellates, which are highly motile and possess four large tufts of eukaryotic agella (Fig. 1), did not yield any evidence for motility of the attached bacteria, and a motility symbiosis seems unlikely. It is more feasible that the interactions between epibionts and hosts are of a metabolic nature. Many bacteria among the Bacteroidales are polysaccharide-fermenting anaerobes, some of them producing cellulases and other bredegrading enzymes, which might complement enzyme activities lacking in the host. Although the large phylogenetic distance to the closest cultivated relative (<85 % sequence identity) allows no safe predictions of the physiological properties of the epibionts, the epibionts might benet from the reduced products of the agellates fermentative metabolism. Again, nothing is known about the fermentation products of Staurojoenina species, but there is circumstantial evidence that the agellates in the hindgut of Reticulitremes avipes form lactate as a major product (Tholen & Brune, 2000). The possibility of a cross feeding of lactate between lactic-acid bacteria and a Bacteroides isolate from this termite has been previously demonstrated (Schultz & Breznak, 1979). Another equally plausible function of the epibionts of benet to the agellate host might lie in the observation that phagocytized cells of the epibionts were regularly encountered in digestive vacuoles (see Fig. 1D). Since wood is an extremely nitrogen-poor diet, it may be that the protein-rich bacteria on the cell membrane are an excellent nitrogen source for the host. Although it is not known whether the intestinal agellates require an organic nitrogen source, the epibionts are likely to assimilate ammonia from the gut uid, and might even x dinitrogen (Breznak, 2000). Obviously, more work is necessary to understand the nature of this symbiosis and the respective functions of the symbiotic partners.

Description of Candidatus Vestibaculum illigatum Vestibaculum illigatum (Ves.ti.bacu.lum. L. fem. n. vestis the covering for the body, clothing, L. neut. n. baculum a (walking) stick, N.L. neut. n. vestibaculum a stick-shaped
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Dolan, M. & Margulis, L. (1997). Staurojoenina and other symbionts

part of the body cover, L. perf. part. pass. illigatum bound, fastened, attached). Rod-shaped bacterium of constant diameter (0?3 mm) and variable length (2?56 mm). Gram-negative cell-wall structure with outer membrane. Colonizes the cell surface of Staurojoenina sp., to which it is connected by electrondense extracellular material. Basis of assignment: 16S rRNA gene sequence (accession number AY540335), hybridization with 16S rRNA-targeted oligonucleotide probe (59CCG GTG TGG GGG ACC TTC-39). Source: epibiont of Staurojoenina agellates from the gut of the termite Neotermes cubanus (Freytaud); so far uncultured.

in Neotermes from San Salvador Island, Bahamas. Symbiosis 22, 229239.

Edwards, U., Rogall, T., Blocker, H., Emde, M. & Bottger, E. C. (1989). Isolation and direct complete nucleotide determination of

entire genes. Characterization of a gene coding for 16S ribosomal RNA. Nucleic Acids Res 17, 78437853.
Goss, S. H. & Gunderson, J. H. (2000). Identity of an ectosymbiont

of a devescovinid. Abstr. 100th Gen. Meet. Am. Soc. Microbiol. 2000, abstr. N-149.
Henckel, T., Friedrich, M. & Conrad, R. (1999). Molecular analyses of

the methane-oxidizing microbial community in rice eld soil by targeting the genes of the 16S rRNA, particulate methane monooxygenase, and methanol dehydrogenase. Appl Environ Microbiol 65, 19801990.
Hongoh, Y., Ohkuma, M. & Kudo, T. (2003). Molecular analysis of

ACKNOWLEDGEMENTS
We thank Dr I. Hrdy, Praha, for supplying the termites and Dr J. Krecek, Fort Lauderdale, and Dr K. Krishna, New York, for species identication.

bacterial microbiota in the gut of the termite Reticulitermes speratus (Isoptera, Rhinotermitidae). FEMS Microbiol Ecol 44, 231242.
Hungate, R. E. (1955). Mutualistic intestinal protists. In Biochemistry

and Physiology of Protists, vol. 2, pp. 159199. Edited by S. H. Hutner & A. Lwoff. New York: Academic Press.
Iida, T., Ohkuma, M., Ohtoko, K. & Kudo, T. (2000). Symbiotic

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